Polyampholytes for delivering polyions to a cell

ABSTRACT

An polyampholyte is utilized in a condensed polynucleotide complex for purposes of nucleic acid delivery to a cell. The complex can be formed with an appropriate amount of positive and/or negative charge such that the resulting complex can be delivered to the extravascular space and may be further delivered to a cell.

This Application is a Continuation-In-Part of Ser. No. 09/753,990, nowU.S. Pat. No. 6,383,811, filed on Jan. 2, 2001.

FIELD OF THE INVENTION

The invention relates to compounds and methods for use in biologicsystems. More particularly, polyions are utilized for modifying thecharge (“recharging”) of particles, such as molecules, polymers, nucleicacids and genes for delivery to cells.

BACKGROUND

Polymers are used for drug delivery for a variety of therapeuticpurposes. Polymers have also been used in research for delivery ofnucleic acids (polynucleotides and oligonucleotides) to cells, theprocess is one step in reaching a goal of providing therapeuticprocesses (gene therapy). One of the several methods of nucleic aciddelivery to the cells is the use of DNA-polyion complexes. It has beenshown that cationic proteins like histones and protamines or syntheticpolymers like polylysine, polyarginine, polyornithine, DEAE dextran,polybrene, and polyethylenimine may be effective intracellular deliveryagents while small polycations like spermine are ineffective.

In terms of intravenous injection, DNA must cross the endothelialbarrier and reach the parenchymal cells of interest. The largestendothelia fenestrae (holes in the endothelial barrier) occur in theliver and have an average diameter from 75-150 nm. The trans-epithelialpores in other organs are much smaller, for example, muscle endotheliumcan be described as a structure which has a large number of small poreswith a radius of 4 nm, and a very low number of large pores with aradius of 20-30 nm. The size of the DNA complexes is also important forthe cellular uptake process. After binding to the target cells theDNA-polycation complex should be taken up by endocytosis.

Applicants have provided a process for delivering a compound across theendothelial barrier to the extravascular space and then to a cell.

SUMMARY

Described in a preferred embodiment is a process for enhancing deliveryof a polyion to a cell, comprising the formation of a complex ofpolyampholyte and polyion. Then, delivering the complex into a cell.

In another preferred embodiment, we describe a process for extravasationof a complex. The process comprises the formation of a complex ofpolyampholyte and polyion. Then, inserting the complex into a vessel anddelivering the complex to an extravascular space.

Reference is now made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 DNA interactions with pC-pA block polyampholyte: binding (pA lowcharge density) and replacement (pA high charge density).

FIG. 2 Isolation of lPEI-pGlu polyampholyte using Example 1 reactionmixture.

FIG. 3 Transfection of HUH7 cells using lPEI-pGlu polyampholyte usingExample 1 reaction mixture.

FIG. 4 Transfection of HUH7 cells using lPEI-pMAA polyampholyte usingExample 1 reaction mixture.

FIG. 5 Sepharose 4B-CL chromatography of rhodamine-labeled pGlu andlPEI-pGlu polyampholyte.

FIG. 6 Luciferase expression in HUH7 cells in vitro in 100% bovine serumaided by covalent lPEI-pGlu polyampholyte as compared to lPEI-pGlunon-covalent mixture and lPEI alone.

DETAILED DESCRIPTION

Polyampholytes are copolyelectrolytes containing both polycations andpolyanions in the same polymer. In aqueous solutions polyampholytes areknown to precipitate near the isoelectric point and form micelle-likestructures (globules) at the excess of either charge. Such globulesmaintain tendency to bind other charged macromolecules and particles(see R R Netz, J F Joanny, Macromolecules, 31, 5123-5141 (1998)).

In provisional application Ser. No. 60/093,153 we described genetransfer activity which can be substantially increased by addingpolyanions to preformed DNA/polycation complexes(i.e. recharging). Weconfirmed the same phenomenon for cationic lipids (provisionalapplication Ser. NO. 60/150,160).

In this application we extend this principle into situations whereDNA-binding polycation and polyanion are covalently linked into onepolymer. Polyanions (polyanion=pA; polycation=pC) of higher chargedensity can displace DNA from its complex with polycation while pAs withlower charge density form triple complexes in which the complexes have anegative surface charge (Y Xu, F C Szoka, Jr., Biochemistry, 35,5616-5623, (1996), V S Trubetskoy, A Loomis, J E Hagstrom, V G Budker, JA Wolff, Nucleic Acids Res. 27, 3090-3095 (1999)). Similarly, one canexpect formation of DNA/polyampholyte complex in situations where apolyanion block ionically attached to a polyampholyte possesses a chargedensity lower than the charge density of the DNA molecule; A DNAmolecule will be released from a complex with a polyampholyte when apolyanion block has a charge density higher than the DNA molecule (seeFIG. 1). In the latter case, an internal pA-pC salt is formed.

It has previously been demonstrated that binding of negatively-chargedserum components can significantly decrease gene transfer efficacy ofDNA/polycation (DNA/pC) complexes in vivo (Vitiello L, Bockhold K, JoshiP B, Worton P B, Gene Therapy 5, 1306-13 (1998); Ross P C, Hui S W, GeneTherapy 6, 651-659 (1999). We have found that addition of polyanions tothe point of near complex charge reversal drastically increases theefficacy of gene transfer mediated by DNA/pC complex upon i/vadministration in mice (Provisional application Ser. No. 60/093,153).This improvement takes place due to protecting effect of pA which issituated as an outside shell on the triple complex and functions byinhibiting interactions of the complexes with serum proteins. We believethat gene transfer increase observed with DNA/polyampholyte complexes isbased on the same phenomenon. The polyanion portion of polyampholytebeing displaced from DNA/pC interaction can form an outside “shell” ofnegative charge and protect the complex from inhibiting interactionswith serum proteins. The charge density of the pA is of primaryimportance. The higher charge density, the more effective is theprotective effect against serum proteins.

In some cases a polyanionic block may be a natural protein or peptideused for cell targeting or other function. A polyanionic block canprovide other functions too: for example, poly(propylacrylic acid) isknown for pH-dependent membrane-disruptive function (Murthy N, RobichaudJ R, Tirrell D A, Stayton P S, Hoffman A S, Controlled Release (1999)61:137-43).

To demonstrate the principle we synthesized two block polyampholytes oflinear polyethyleneimine (lPEI) with 1) polymethacrylic acid (lPEI-pMAA,high charge density pA) and polyglutamic acid (lPEI-pGlu, low chargedensity pA) and prepared complexes with plasmid DNA (pCIluc). We showthat a covalent complex between pC and pA can substantially enhance genetransfer activity when compared to a non-polyampholyte mixture. Wefurther describe the phenomena in the examples section of thisapplication.

In this specification, the use of the term polyanion may refer to theanionic portion of the polyampholyte and the term polycation may referto the cationic portion of the polyampholyte. Abbreviations:Poly-L-Lysine (PLL), succinic anhydride-PLL (SPLL), polymethacrylicacid, pMAA and polyaspartic acid, pAsp

Polymers

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer.

Step Polymerization: In step polymerization, the polymerization occursin a stepwise fashion. Polymer growth occurs by reaction betweenmonomers, oligomers and polymers. No initiator is needed since there isthe same reaction throughout and there is no termination step so thatthe end groups are still reactive. The polymerization rate decreases asthe functional groups are consumed.

Typically, step polymerization is done either of two different ways. Oneway, the monomer has both reactive functional groups (A and B) in thesame molecule so thatA-B yields -(A-B)-

Or the other approach is to have two difunctional monomers.A-A+B-B yields -(A-A-B-B)-

Generally, these reactions can involve acylation or alkylation.Acylation is defined as the introduction of an acyl group (—COR) onto amolecule. Alkylation is defined as the introduction of an alkyl grouponto a molecule.

“If functional group A is an amine then B can be (but not restricted to)an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide,sulfonyl chloride,aldehyde (including formaldehyde and glutaraldehyde),ketone, epoxide, carbonate, imidoester, carboxylate activated with acarbodiimide, alkylphosphate, arylhalides (difluoro-dinitrobenzene),anhydride, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonylimidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium. In other termswhen function A is an amine then function B can be acylating oralkylating agent or amination agent.

If functional group A is a sulfhydryl then function B can be (but notrestricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoicacid{TNB} derivatives).

If functional group A is carboxylate then function B can be (but notrestricted to) adiazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

If functional group A is an hydroxyl then function B can be (but notrestricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N,N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used. Iffunctional group A is an aldehyde or ketone then function B can be (butnot restricted to) an hydrazine, hydrazide derivative, amine (to form aSchiff Base that may or may not be reduced by reducing agents such asNaCNBH3) or hydroxyl compound to form a ketal or acetal.

Yet another approach is to have one bifunctional monomer so that A-Aplus another agent yields -(A-A)-. If function A is a sulfhydryl groupthen it can be converted to disulfide bonds by oxidizing agents such asiodine (I2 ) or NaIO4 (sodium periodate), or oxygen (O2). Function A canalso be an amine that is converted to a sulfhydryl group by reactionwith 2-Iminothiolate (Traut's reagent) which then undergoes oxidationand disulfide formation. Disulfide derivatives (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) can also beused to catalyze disulfide bond formation. Functional group A or B inany of the above examples could also be a photoreactive group such asaryl azide (including halogenated aryl azide), diazo, benzophenone,alkyne or diazirine derivative.

Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yieldchemical bonds that are described as amide, amidine, disulfide, ethers,esters, enamine, imine, urea, isothiourea, isourea, sulfonamide,carbamate, alkylamine bond (secondaryamine), carbon-nitrogen singlebonds in which the carbon contains a hydroxyl group, thioether, diol,hydrazone, diazo, or sulfone”.

If functional group A is an aldehyde or ketone then function B can be(but not restricted to) an hydrazine, hydrazide derivative, amine (toform a Schiff Base that may or may not be reduced by reducing agentssuch as NaCNBH3) or hydroxyl compound to form a ketal or acetal.

Yet another approach is to have one difunctional monomer so thatA-A plus another agent yields -(A-A)-.

If function A is a sulfhydryl group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I2) or NaIO4 (sodiumperiodate), or oxygen (O2). Function A can also be an amine that isconverted to a sulfhydryl group by reaction with 2-iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid{TNB} derivatives) can also be used tocatalyze disulfide bond formation.

Functional group A or B in any of the above examples could also be aphotoreactive group such as aryl azides, halogenated aryl azides, diazo,benzophenones, alkynes or diazirine derivatives.

Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yieldchemical bonds that are described as amide, amidine, disulfide, ethers,esters, enamine, urea, isothiourea, isourea, sulfonamide, carbamate,carbon-nitrogen double bond (imine), alkylamine bond (secondary amine),carbon-nitrogen single bonds in which the carbon contains a hydroxylgroup, thio-ether, diol, hydrazone, diazo, or sulfone.

Chain Polymerization: In chain-reaction polymerization growth of thepolymer occurs by successive addition of monomer units to limited numberof growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain-terminating step. Thepolymerization rate remains constant until the monomer is depleted.

Monomers containing vinyl, acrylate, methacrylate, acrylamide,methaacrylamide groups can undergo chain reaction which can be radical,anionic, or cationic. Chain polymerization can also be accomplished bycycle or ring opening polymerization. Several different types of freeradical initiatiors could be used that include peroxides, hydroxyperoxides, and azo compounds such as 2,2′-Azobis(-amidinopropane)dihydrochloride (AAP). A compound is a material made up of two or moreelements.

Types of Monomers: A wide variety of monomers can be used in thepolymerization processes. These include positive charged organicmonomers such as amines, imidine, guanidine, imine, hydroxylamine,hydrozyine, heterocycles (like imidazole, pyridine, morpholine,pyrimidine, or pyrene. The amines could be pH-sensitive in that the pKaof the amine is within the physiologic range of 4 to 8. Specific aminesinclude spermine, spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine(AEPD), and 3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

Monomers can also be hydrophobic, hydrophilic or amphipathic.Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties carbohydrates;polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bond. Hydrocarbons are hydrophobicgroups. Monomers can also be intercalating agents such as acridine,thiazole organge, or ethidium bromide.

Other Components of the Monomers and Polymers: The polymers have othergroups that increase their utility. These groups can be incorporatedinto monomers prior to polymer formation or attached to the polymerafter its formation. These groups include: Targeting Groups—such groupsare used for targeting the polymer-nucleic acid complexes to specificcells or tissues. Examples of such targeting agents include agents thattarget to the asialoglycoprotein receptor by using asiologlycoproteinsor galactose residues. Other proteins such as insulin, EGF, ortransferrin can be used for targeting. Protein refers to a molecule madeup of 2 or more amino acid residues connected one to another as in apolypeptide. The amino acids may be naturally occurring or synthetic.Peptides that include the RGD sequence can be used to target many cells.Peptide refers to a linear series of amino acid residues connected toone another by peptide bonds between the alpha-amino group and carboxylgroup of contiguous amino acid residues. Chemical groups that react withsulfhydryl or disulfide groups on cells can also be used to target manytypes of cells. Folate and other vitamins can also be used fortargeting. Other targeting groups include molecules that interact withmembranes such as fatty acids, cholesterol, dansyl compounds, andamphotericin derivatives.

After interaction of the supramolecular complexes with the cell, othertargeting groups can be used to increase the delivery of the drug ornucleic acid to certain parts of the cell. For example, agents can beused to disrupt endosomes and a nuclear localizing signal (NLS) can beused to target the nucleus.

A variety of ligands have been used to target drugs and genes to cellsand to specific cellular receptors. The ligand may seek a target withinthe cell membrane, on the cell membrane or near a cell. Binding ofligands to receptors typically initiates endocytosis. Ligands could alsobe used for DNA delivery that bind to receptors that are notendocytosed. For example peptides containing RGD peptide sequence thatbind integrin receptor could be used. In addition viral proteins couldbe used to bind the complex to cells. Lipids and steroids could be usedto directly insert a complex into cellular membranes.

The polymers can also contain cleavable groups within themselves. Whenattached to the targeting group, cleavage leads to reduce interactionbetween the complex and the receptor for the targeting group. Cleavablegroups include but are not restricted to disulfide bonds, diols, diazobonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enolesters, enamines and imines.

Reporter or marker molecules are compounds that can be easily detected.Typically they are fluorescent compounds such as fluorescein, rhodamine,texas red, CY-5, CY-3 or dansyl compounds. They can be molecules thatcan be detected by UV or visible spectroscopy or by antibodyinteractions or by electron spin resonance. Biotin is another reportermolecule that can be detected by labeled avidin. Biotin could also beused to attach targeting groups.

A polycation is a polymer containing a net positive charge, for examplepoly-L-lysine hydrobromide. The polycation can contain monomer unitsthat are charge positive, charge neutral, or charge negative, however,the net charge of the polymer must be positive. A polycation also canmean a non-polymeric molecule that contains two or more positivecharges. A polyanion is a polymer containing a net negative charge, forexample polyglutamic acid. The polyanion can contain monomer units thatare charge negative, charge neutral, or charge positive, however, thenet charge on the polymer must be negative. A polyanion can also mean anon-polymeric molecule that contains two or more negative charges. Theterm polyion includes polycation, polyanion, zwitterionic polymers, andneutral polymers. The term zwitterionic refers to the product (salt) ofthe reaction between an acidic group and a basic group that are part ofthe same molecule. Salts are ionic compounds that dissociate intocations and anions when dissolved in solution. Salts increase the ionicstrength of a solution, and consequently decrease interactions betweennucleic acids with other cations. A charged polymer is a polymer thatcontains residues, monomers, groups, or parts with a positive ornegative charge and whose net charge can be neutral, positive, ornegative.

Signals

In a preferred embodiment, a chemical reaction can be used to attach asignal to a nucleic acid complex. The signal is defined in thisspecification as a molecule that modifies the nucleic acid complex andcan direct it to a cell location (such as tissue cells) or location in acell (such as the nucleus) either in culture or in a whole organism. Bymodifying the cellular or tissue location of the foreign gene, theexpression of the foreign gene can be enhanced.

The signal can be a protein, peptide, lipid, steroid, sugar,carbohydrate, nucleic acid or synthetic compound. The signals enhancecellular binding to receptors, cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

Signals that enhance release from intracellular compartments (releasingsignals) can cause DNA release from intracellular compartments such asendosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmicreticulum, Golgi apparatus, trans Golgi network (TGN), and sarcoplasmicreticulum. Release includes movement out of an intracellular compartmentinto cytoplasm or into an organelle such as the nucleus. Releasingsignals include chemicals such as chloroquine, bafilomycin or BrefeldinAl and the ER-retaining signal (KDEL sequence; SEQ ID 1), viralcomponents such as influenza virus hemagglutinin subunit HA-2 peptidesand other types of amphipathic peptides.

Cellular receptor signals are any signal that enhances the associationof the gene or particle with a cell. This can be accomplished by eitherincreasing the binding of the gene to the cell surface and/or itsassociation with an intracellular compartment, for example: ligands thatenhance endocytosis by enhancing binding the cell surface. This includesagents that target to the asialoglycoprotein receptor by usingasiologlycoproteins or galactose residues. Other proteins such asinsulin, EGF, or transferrin can be used for targeting. Peptides thatinclude the RGD sequence can be used to target many cells. Chemicalgroups that react with sulfhydryl or disulfide groups on cells can alsobe used to target many types of cells. Folate and other vitamins canalso be used for targeting. Other targeting groups include moleculesthat interact with membranes such as lipids fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives. In addition viralproteins could be used to bind cells.

Other Definitions:

Extravascular means outside of a vessel such as a blood vessel.Extravascular space means an area outside of a vessel. Space may containbiological matter such as cells and does not imply empty space.

Extravasation means the escape of material such as compounds andcomplexes from the vessel into which it is introduced into theparenchymal tissue or body cavity.

The process of delivering a polynucleotide to a cell has been commonlytermed “transfection” or the process of “transfecting” and also it hasbeen termed “transformation”. The polynucleotide could be used toproduce a change in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The polynucleotides orgenetic material being delivered are generally mixed with transfectionreagents prior to delivery.

The polyampholyte complex is a complex having the potential to reactwith biological components. More particularly, polyampholyte complexesutilized in this specification are designed to change the naturalprocesses associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a polyampholyte complex. Inthis specification, the cellular production of, or inhibition of amaterial, such as a protein, caused by a human assisting a molecule toan in vivo cell is an example of a delivered biologically activecompound. Pharmaceuticals, proteins, peptides, polypeptides, hormones,cytokines, antigens, viruses, oligonucleotides, and nucleic acids areexamples that can be components of polyampholyte complexes.

The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). DNA may be in the form of anti-sense, plasmid DNA, parts ofa plasmid DNA, vectors (P1, PAC, BAC, YAC, artificial chromosomes),expression cassettes, chimeric sequences, chromosomal DNA, orderivatives of these groups. RNA may be in the form of oligonucleotideRNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomalRNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimericsequences, or derivatives of these groups. “Anti-sense” is apolynucleotide that interferes with the function of DNA and/or RNA. Thismay result in suppression of expression. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones, nucleotides, or bases. These include PNAs (peptide nucleicacids), phosphothionates, and other variants of the phosphate backboneof native nucleic acids. In addition, DNA and RNA may be single, double,triple, or quadruple stranded. “Expression cassette” refers to a naturalor recombinantly produced polynucleotide molecule which is capable ofexpressing protein(s). A DNA expression cassette typically includes apromoter (allowing transcription initiation), and a sequence encodingone or more proteins. Optionally, the expression cassette may includetranscriptional enhancers, non-coding sequences, splicing signals,transcription termination signals, and polyadenylation signals. An RNAexpression cassette typically includes a translation initiation codon(allowing translation initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may include translationtermination signals, a polyadenosine sequence, internal ribosome entrysites (IRES), and non-coding sequences.

The term “naked polynucleotides” indicates that the polynucleotides arenot associated with a transfection reagent or other delivery vehiclethat is required for the polynucleotide to be delivered to the cardiacmuscle cell. A “transfection reagent” is a compound or compounds used inthe prior art that bind(s) to or complex(es) with oligonucleotides andpolynucleotides, and mediates their entry into cells. The transfectionreagent also mediates the binding and internalization ofoligonucleotides and polynucleotides into cells. Examples oftransfection reagents include cationic liposomes and lipids, polyamines,calcium phosphate precipitates, histone proteins, polyethylenimine, andpolylysine complexes. It has been shown that cationic proteins likehistones and protamines, or synthetic polymers like polylysine,polyarginine, polyornithine, DEAE dextran, polybrene, andpolyethylenimine may be effective intracellular delivery agents, whilesmall polycations like spermine may be ineffective. Typically, thetransfection reagent has a net positive charge that binds to theoligonucleotide's or polynucleotide's negative charge. The transfectionreagent mediates binding of oligonucleotides and polynucleotides tocells via its positive charge (that binds to the cell membrane'snegative charge) or via ligands that bind to receptors in the cell. Forexample, cationic liposomes or polylysine complexes have net positivecharges that enable them to bind to DNA or RNA. Polyethylenimine, whichfacilitates gene expression without additional treatments, probablydisrupts endosomal function itself.

Other vehicles are also used, in the prior art, to transfer genes intocells. These include complexing the polynucleotides on particles thatare then accelerated into the cell. This is termed “biolistic” or “gun”techniques. Other methods include “electroporation,” in which a deviceis used to give an electric charge to cells. The charge increases thepermeability of the cell.

Charge density is the term used to describe the electrical charge perunit area, for example, on a polymer.

Ionic (electrostatic) interactions are the non-covalent association oftwo or more substances due to attractive forces between positive andnegative charges, or partial positive and partial negative charges.

Condensed Nucleic Acids: Condensing a polymer means decreasing thevolume that the polymer occupies. An example of condensing nucleic acidis the condensation of DNA that occurs in cells. The DNA from a humancell is approximately one meter in length but is condensed to fit in acell nucleus that has a diameter of approximately 10 microns. The cellscondense (or compacts) DNA by a series of packaging mechanisms involvingthe histones and other chromosomal proteins to form nucleosomes andchromatin. The DNA within these structures is rendered partiallyresistant to nuclease DNase) action. The process of condensing polymerscan be used for delivering them into cells of an organism.

A delivered polymer can stay within the cytoplasm or nucleus apart fromthe endogenous genetic material. Alternatively, the polymer couldrecombine (become a part of) the endogenous genetic material. Forexample, DNA can insert into chromosomal DNA by either homologous ornon-homologous recombination.

Condensed nucleic acids may be delivered intravasculary, intrarterially,intravenously, orally, intraduodenaly, via the jejunum (or ileum orcolon), rectally, transdermally, subcutaneously, intramuscularly,intraperitoneally, intraparenterally, via direct injections into tissuessuch as the liver, lung, heart, muscle, spleen, pancreas, brain(including intraventricular), spinal cord, ganglion, lymph nodes,lymphatic system, adipose tissues, thyroid tissue, adrenal glands,kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors,eye retina, via the bile duct, or via mucosal membranes such as in themouth, nose, throat, vagina or rectum or into ducts of the salivary orother exocrine glands. “Delivered” means that the polynucleotide becomesassociated with the cell. The polynucleotide can be on the membrane ofthe cell or inside the cytoplasm, nucleus, or other organelle of thecell.

An intravascular route of administration enables a polymer orpolynucleotide to be delivered to cells more evenly distributed and moreefficiently expressed than direct injections. Intravascular herein meanswithin a tubular structure called a vessel that is connected to a tissueor organ within the body. Within the cavity of the tubular structure, abodily fluid flows to or from the body part. Examples of bodily fluidinclude blood, lymphatic fluid, or bile. Examples of vessels includearteries, arterioles, capillaries, venules, sinusoids, veins,lymphatics, and bile ducts. The intravascular route includes deliverythrough the blood vessels such as an artery or a vein.

An administration route involving the mucosal membranes is meant toinclude nasal, bronchial, inhalation into the lungs, or via the eyes.

Recharging Condensed Nucleic Acids

Polyions for gene therapy and gene therapy research can involve anionicsystems as well as charge neutral or charge-positive systems. The ionicpolymer can be utilized in “recharging” (another layer having adifferent charge) the condensed polynucleotide complex. The resultingrecharged complex can be formed with an appropriate amount of chargesuch that the resulting complex has a net negative, positive or neutralcharge. The interaction between the polycation and the polyanion can beionic, can involve the ionic interaction of the two polymer layers withshared cations, or can be crosslinked between cationic and anionic siteswith a crosslinking system (including cleavable crosslinking systems,such as those containing disulfide bonds). The interaction between thecharges located on the two polymer layers can be influenced with the useof added ions to the system. With the appropriate choice of ion, thelayers can be made to disassociate from one another as the ion diffusesfrom the complex into the cell in which the concentration of the ion islow (use of an ion gradient).

Electrostatic complexes between water-soluble polyelectrolytes have beenstudied widely in recently ears. Complexes containing DNA as apolyanionic constituent only recently came to the attention because oftheir potential use in gene therapy applications such as non-viral genetransfer preparations (polyplexes) for particle delivery to a cell.Strong polyelectrolytes, polyanion/polycation complexes, are usuallyformed at a 1:1 charge stoichiometrically. A charge ratio 1:1 complexbetween DNA and Poly-L-Lysine (PLL) also has been demonstrated in theprior art.

Polyanions effectively enhance the gene delivery/gene expressioncapabilities of all major classes of polycation gene delivery reagents.In that regard, we disclose the formation of negatively charged tertiarycomplexes containing nucleic acid, PLL, and succinic anhydride-PLL(SPLL) complexes. SPLL is added to a cationic nucleic acid/PLL complexin solution. Nucleic acid at the core of such complexes remainscondensed, in the form of particles ˜50 nm in diameter. DNA and PLLbinds SPLL in 1:1:1 complex with SPLL providing a net negative charge tothe entire complex. Such small negatively charged particles are usefulfor non-viral gene transfer applications.

One of the advantages that flow from recharging DNA particles isreducing their non-specific interactions with cells and serum proteins((Wolfert et al. Hum. Gene Therapy 7:2123-2133 (1996); Dash et al., GeneTherapy 6:643-650 (1999); Plank et al., Hum. Gene Ther. 7:1437-1446(1996); Ogris et al., Gene Therapy 6:595-605 (1999); Schacht et al.Brit. Patent Application 9623051.1 (1996))

A wide a variety of polyanions can be used to recharge theDNA/polycation particles. They include (but not restricted to): Anywater-soluble polyanion can be used for recharging purposes includingsuccinylated PLL, succinylated PEI (branched), polyglutamic acid,polyaspartic acid, polyacrylic acid, polymethacrylic acid,polyethylacrylic acid, polypropylacrylic acid, polybutylacrylic acid,polymaleic acid, dextran sulfate, heparin, hyaluronic acid,polysulfates, polysulfonates, polyvinyl phosphoric acid, polyvinylphosphonic acid, copolymers of polymaleic acid, polyhydroxybutyric acid,acidic polycarbohydrates, DNA, RNA, negatively charged proteins,pegylated derivatives of above polyanions, pegylated derivativescarrying specific ligands, block and graft copolymers of polyanions andany hydrophilic polymers (PEG, poly(vinylpyrrolidone), poly(acrylamide),etc).

DNA condensation assays based on the effect of concentration-dependentself-quenching of covalently-bound fluorophores upon DNA collapseindicated essentially the same phenomenon described in the prior art.Polyanions with high charge density (polymethacrylic acid, pMAA andpolyaspartic acid, pAsp) were able to decondense DNA prior to those thatcomplexed with PLL while polyanions with lower charge density(polyglutamic acid, pGlu, SPLL) failed to decondense DNA. Together withz-potential measurements, these data represent support for the presenceof negatively charged condensed DNA particles. These particles areapproximately 50 nm in diameter in low salt buffer as measured by atomicforce microscopy which revealed particles of spheroid morphology. Thisplaces them very close in size to binary DNA/PLL particles. Particlesprepared using various pC/pA polyampholytes can be used to form similarcondensed DNA particles.

In another preferred embodiment, the polyanion can be covalentlyattached to the polycation using a variety of chemical reactions withoutthe use of crosslinker. The polyanion can contain reactive groups thatcovalently attach to groups on the polycation. This results in theformation of a polyampholyte The types of reactions are similar to thosediscussed above in the section on step polymerization.

In another preferred embodiment the attachment of the recharged complexcan be enhanced by using chelators and crown ethers, preferablypolymeric.

In one preferred embodiment the DNA/polycation complexes are initiallyformed by adding only a small excess of polycation to nucleic acid (incharge ratio which is defined as ratio of polycation total charge topolyanion total charge at given pH). The charge ratio of polycation tonucleic acid charge could be less than 2, less than 1.7, less than 1.5or even less than 1.3. This would be preferably done in low ionicstrength solution so as to avoid the complexes from flocculation. Lowionic strength solution means solution with total monovalent saltconcentration less than 50 mM. Then the polyanion is added to themixture and only a small amount of “blank” particles are formed. “Blank”particles are particles that contain only polycation and polyanion andno nucleic acid.

In another preferred embodiment, the polycation is added to the nucleicacid in charge excess but the excess polycation that is not in complexwith the nuclei acid is removed by purification. Purification meansremoving of charged polymer using centrifugation, dialysis,chromatography, electrophoresis, precipitation, extraction.

Yet in another preferred embodiment a ultracentrifugation procedure(termed “centrifugation step”) is used to reduce the amount of excesspolycation, polyanion, or “blank” particles. The method is based on thephenomenon that only dense DNA-containing particles can be centrifugedthrough 10% sucrose solution at 25,000 g. After centrifugation purifiedcomplex is at the bottom of the tube while excess of polyanion and“blank” particles stay on top. In modification of this experiment 40%solution of metrizamide can be used as a cushion to collect purifiedDNA/polycation/polyanion complex on the boundary for easy retrieval.

The attachment of the polyanion to the DNA/polycation complex enhancestability but can also enable a ligand or signal to be attached to theDNA particle. This is accomplished by attaching the ligand or signal tothe polyanion which in turn is attached to the DNA particle. A dialysisstep or centifugation step can be used to reduce the amount of freepolyanion containing a ligand or signal that is in solution and notcomplexed with the DNA particle. One approach is to replace the free,uncomplexed polyanion containing a ligand or signal with free polyanionthat does not contain a ligand or signal.

Yet in another preferred embodiment a polyanion used for charge reversalis modified with neutral hydrophilic polymer for steric stabilization ofthe whole complex. The complex formation of DNA with pegylatedpolycations results in substantial stabilization of the complexestowards salt- and serum-induced flocculation (Wolfert et al. Hum. GeneTherapy 7:2123-2133 (1996), Ogris et al., Gene Therapy 6:595-605 (1999).We have demonstrated that modification of polyanion in triple complexalso significantly enhances salt and serum stability.

In another preferred embodiment a polyanion used for charge reversal iscleavable. One can imagine two ways to design a cleavable polyion: 1. Apolyion cleavable in backbone, 2. A polyion cleavable in side chain.First scenario would comprise monomers linked by labile bonds such asdisulfide, diols, diazo, ester, sulfone, acetal, ketal, enol ether, enolester, imine and enamine bonds. Second scenario would involve reactivegroups (i.e. electrophiles and nucleophiles) in close proximity so thatreaction between them is rapid. Examples include having corboxylic acidderivatives (acids, esters and amides) and alcohols, thiols, carboxylicacids or amines in the same molecule reacting together to make esters,thiol esters, anhydrides or amides. In one specific preferred embodimentthe polyion contains an ester acid such as citraconnic acid, ordimethylmaleyl acid that is connected to a carboxylic, alcohol, or aminegroup on the polyion.

Cleavable means that a chemical bond between atoms is broken. Labilealso means that a chemical bond between atoms is breakable. Crosslinkingrefers to the chemical attachment of two or more molecules with abifunctional reagent. A bifunctional reagent is a molecule with tworeactive ends. The reactive ends can be identical as in ahomobifunctional molecule, or different as in a heterobifunctionalmolecule.

EXAMPLES Example 1

Synthesis of lPEI-pMAA and lPEI-pGlu complexes.

The following polyions were used for the reaction: lPEI (Mw=25 kDa,Polysciences), pMAA (Mw=9.5 kDa, Aldrich), pGlu (Mw=49 kDa, Sigma). Foranalytical purposes pAs covalently labeled withrhodamine-ethylenediamine (Molecular Probes) were used for thesereactions (degree of carboxy group modification<2%). Absorbance of thepAs was used to trace pAs and conjugates during size exclusionchromatography. PMAA (or pGlu, 1 mg in 100 μL water) was activated inthe presence of water-soluble carbidiimide (EDC, 100 μg) andN-hydroxysulfosuccinimide (100 μg) for 10 min at pH 5.5. Activated pMAAwas added to the solution of lPEI (2 mg in 200 μL of 25 mM HEPES, pH8.0) and incubated for 1 hr at room temperature.

Example 2

Separation of lPEI-pMAA and lPEI-pGlu reaction mixtures using Sepharose4B-CL column in 1.5 M NaCl.

After the reaction completion equal volume of 3 M NaCl solution wasadded to the part of the reaction mixture. This part (0.5 ml) was passesthrough the Sepharose 4B-CL column (1×25 cm) equlibrated in 1.5 M NaCl.Volume of the fractions collected was 1 ml. Rhodamine fluorescence wasmeasured in each fraction. Linear PEI was measured using fluorescaminereaction. The amount of polyampholyte in the lPEI-pGlu reaction mixtureis about 50% (see FIG. 2).

Example 3

HUH7 mouse liver cell transfection using DNA/lPEI-pA polyampholytemixtures.

Part of the polyampholyte reaction mixtures lPEI-pMAA and lPEI-pGlu wereused to transfect HUH7 cells in culture. Non-covalent mixtures of lPEIwith pMAA and pGlu mixed in the same ratios as for conjugates were usedas the controls. Luciferase-encoded plasmid pCIluc (2 μg per 35 mm well)was used for transfection in OPTIMEM (cell medium) and OPTIMEMsupplemented with 10% bovine serum. Amount of polyampholyte added isindicated on the basis of lPEI content. Results of this experiment areshown on FIGS. 3 and 4. There is a strong enhancement of transfectionfor polyampholytes in case of weaker pA conjugate (lPEI-pGlu, FIG. 3)and almost no difference in transfection abilities of conjugates andmixtures for stronger pA (lPEI-pMAA, FIG. 4) in accordance to FIG. 1scheme.

Example 4

Optimized Synthesis of lPEI-pGlu Polyampholyte

Rhodamine-labeled polyglutamic acid (pGlu, 150 uL, 20 mg/ml, titrated topH 5.0) was activated with water-soluble[3′-(dimethylaminopropyl)-3-ethyl]carbodiimide (EDC, 15 ul, 100 mg/ml inDMSO) and sulfosuccinimide (SNHS, 15 um, 100 mg/ml in water) for 10 min.Then linear PEI (lPEI, 150 ul, 20 mg/ml) was added to the mixture, pHwas adjusted to 8.0 and the mixture was allowed to stand for 2 hrs atroom temperature. After this the mixture was passed through Sepharose4B-CL column (1×20 cm) equilibrated with 1.5 M NaCl solution (FIG. 5).Rhodamine fluorescence was measured in each fraction. Fractions 10-14were pooled, dialysed against water and freeze-dried to yield purifiedpolyampholyte.

Example 5

HUH7 mouse liver cell transfection using DNA/lPEI-pA polyampholytemixtures in the presence of 100% serum.

The luciferase encoding plasmid pCILuc was used for in vitro and in vivogene transfer experiments. The DNA/polymer complexes were formed in 5 mMHEPES, 50 mM NaCl, 0.29 M glucose, pH 7.5 at DNA concentration of 50ug/ml. HUH7 mouse liver cells were subconfluently seeded in 12-wellplates. The complexes (1 ug of DNA) were added directly to 1 ml of 100%bovine serum into each well and incubated with cells for 4 hrs. Afterthis step the cells were washed with OPTI-MEM media, supplemented withfresh media and maintained for additional 48 hrs. After this period oftime the cells were harvested, lysed and processed for luciferaseexpression measurements. Non-covalent mixture of lPEI and pGlu as wellas lPEI alone were used a controls in this experiment (FIG. 6). As onecan see, the covalent conjugate of lPEI and pGlu gave significantlyhigher gene transfer activity in high range of polymer/DNA ratios ascompared to controls.

Example 6

Synthesis of Branched PEI (brPEI)—pGlu and brPEI-pAsp Polyampholytes

Polyglutamic acid (pGlu, 2.28 mg in 172 ul of water, pH 5.5) orpolyaspartic acid (pAsp, 2 mg in 172 ul of water) were activated in thepresence of 100 ug of EDC and SNHS each for 10 min at room temperature.BrPEI (4 mg) and 2.5 M Na Cl (0.5 ml) solutions were added to theactivated polyanion. The reaction mixture was allowed to incubate for 5hrs at room temperature. Resulting brPEI-based polyampholytes weredialyzed against water and freeze-dried.

Example 7

In vivo gene transfer activity of DNA/polyampholyte complexes preparedfrom branched PEI.

BrPEI-pGlu and brPEI-pAsp polyampholytes were mixed with DNA atdifferent w/w ratios in 5 mM HEPES, 0.29 M glucose, pH 7.5 at the DNAconcentration of 0.2 mg/ml. The complexes (0.25 ml per animal) wereintravenously injected into mouse tail vein (2 animals per group). Theanimals were sacrificed 24 hrs after injection and the lungs wereprocessed for luciferase activity. The results of in vivo gene transferare presented in Table 1:

TABLE 1 DNA/brPEI- DNA/brPEI- Ratio (w/w) DNA/brPEI pAsp pGlu 1:1 600,non-toxic 88,000, non- 34,000, non- toxic toxic 1:2 All died 600,000,one 3,900,000, died non-toxic 1:3 n/a n/a 4,800,000, one died Luciferaseactivity (RLU) in lungs after intravenous administration ofDNA/brPEI-based polyampholytes in mice. Each animal received 50 ug ofDNA in 0.25 ml of isotonic glucose solution. There were 2 animals pergroup. Survival of all animals in the group marked as non-toxic.

As one can see, complexing DNA with brPEI-based polyampholytes resultsin effective preparations for DNA delivery to parenchymal cells. BrPEIalone is ineffective at low weight ratios and toxic at higher ratios.Covalent conjugation of polyanions results in significant increase ingene transfer efficacy in lungs accompanying with reduction of toxicity.

The foregoing is considered as illustrative only of the principles ofthe invention. Further, since numerous modifications and changes willreadily occur to those skilled in the art, it is not desired to limitthe invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

1. A process for enhancing delivery of a polyion to a cell, comprising:a) forming a complex of polyampholyte having groups with pKa's withinthe physiological range of 4 to 8 and polyion; and, b) delivering thecomplex into a cell.
 2. The process of claim 1 wherein the polyampholytecontains maleamic acid.
 3. The process of claim 2 wherein thepolyampholyte is delivered to a cell in vivo.
 4. A deliverable complex,comprising: a polyion, and a polyampholyte having groups with pKa'swithin the physiological range of 4 to 8 wherein the complex isstructured to enter a mammalian cell.
 5. The complex of claim 4 whereinthe polyampholyte contains a bond that is cleavable by a decrease in pH.6. The complex of claim 5 wherein the polyampholyte contains maleamicacid.
 7. The complex of claim 6 wherein the polyampholyte ismembrane-disruptive upon cleavage.
 8. The complex of claim 7 wherein thepolyion comprises a polynucleotide.
 9. The complex of claim 8 whereinthe polynucleotide comprises an double strand RNA oligonucleotide. 10.The complex of claim 1 wherein the polyampholyte is amphipathic.
 11. Thecomplex of claim 1 wherein the polyampholyte contains targeting groups.12. The complex of claim 11 wherein the targeting group comprises aligand for a cellular receptor.
 13. The complex of claim 12 wherein thecellular receptor consists of an asialoglycoprotein receptor.
 14. Thecomplex of claim 1 wherein the polyampholyte contains one or morepolyethylene glycol groups.